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Effects of SLFN5 knockdown on RCC morphology. (A) 786-0 cells were transiently transfected with control siRNA or SLFN5 siRNA, and after cell lysis, total lysates were resolved by SDS-PAGE and immunoblotted with an anti-SLFN5 antibody or an anti-GAPDH antibody, as indicated. (B) 786-0 cells stably expressing control shRNA-GFP or SLFN5 shRNA-GFP were lysed, and total cell lysates were resolved by SDS-PAGE and immunoblotted with anti-SLFN5 or anti-GAPDH antibodies, as indicated. (C) 786-0 cells were transiently transfected with control (CTRL) siRNA or SLFN5 siRNA. Forty-eight hours later, cells were imaged by phase-contrast microscopy at low (left panels) and high (right panels) magnifications. (D) 786-0 cells seeded onto glass coverslips were transiently transfected with control siRNA or SLFN5 siRNA with Lipofectamine RNAiMAX; at 48 h after transfection, the cells were fixed, permeabilized, and stained. Confocal fluorescence microscopy of focal adhesion and actin cytoskeleton was performed with triple labeling, using tetramethyl rhodamine-isothiocyanate (TRITC)-conjugated phalloidin (lower right panels), antivinculin monoclonal antibody (lower left panels), and 4′,6-diamidino-2-phenylindole (DAPI) (upper right panels). Overlay of fluorescent signals is shown in upper left panels. Images are representative areas of the entire coverslip analyzed. Bars, 50 μm. (E) 786-0 cells stably expressing control shRNA-GFP or SLFN5 shRNA-GFP were seeded for 24 h onto glass coverslips. Cells were then fixed, permeabilized, and stained with Alexa Fluor 568-labeled phalloidin to detect cytoskeletal F-actin by confocal fluorescence microscopy. Representative areas showing the F-actin structure are shown. (F) Diameter distributions of 786-0 control shRNA-GFP and SLFN5 shRNA-GFP cells were measured using the Scepter 2.0 cell counter. Differences in cell diameter are plotted on a bar graph and represent means ± standard errors from three independent counts.

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FIG 4

Knockdown of , , or does not result in loss of stress fibers and reduction in cell size. (A to E) 786-0 cells seeded for 24 h onto glass coverslips were transiently transfected with control siRNA (A), SLFN5 siRNA (B), SLFN11 siRNA (C), SLFN12 siRNA (D), or SLFN13 siRNA (E) using Lipofectamine RNAiMAX; at 48 h after transfection, the cells were fixed, permeabilized and incubated with 4′,6-diamidino-2-phenylindole and Alexa Fluor 568-labeled phalloidin. Overlays of representative fluorescence signals are shown. (F) 786-0 cells were transiently transfected, respectively, with SLFN5, SLFN11, SLFN12, or SLFN13 siRNA or control siRNA, and mRNA expression for different SLFNs was measured by real-time RT-PCR, using a GAPDH primer as a control. Data are expressed as the fold change over controls and represent means ± standard errors of results from three independent experiments.

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FIG 5

SLFN5 knockdown does not affect cell proliferation. (A) Equal numbers of 786-0 control siRNA- and SLFN5 siRNA-transfected cells were cultured for the indicated times. After 1, 2, 4, 5, and 6 days of incubation, cell proliferation was assessed by WST assays. Data are expressed as means ± standard errors of results from three independent experiments. (B) Control shRNA-GFP and SLFN5 shRNA-GFP 786-0 cells were seeded in culture dishes and harvested after 3 or 4 days. Cell counts were obtained using the trypan blue dye exclusion test. Data are expressed as means ± standard errors of results from three independent experiments.

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FIG 6

Effect of SLFN5 overexpression on cell morphology. (A) 786-0 pLVX/tetONE-puro-SLFN5-Myc-Flag tag-transduced cells were treated with doxycycline (Dox) as indicated, and after cell lysis, proteins were resolved by SDS-PAGE and immunoblotted with an anti-SLFN5 antibody and an antitubulin antibody. (B) The same cells as used in panel A were treated with doxycycline for 72 h and then seeded onto 0.2% gelatin-coated glass coverslips. Cells were then fixed, permeabilized, and stained with Alexa Fluor 568-labeled phalloidin and 4′,6-diamidino-2-phenylindole (DAPI) to detect cytoskeletal F-actin and nuclei by confocal fluorescence microscopy. Representative areas showing the F-actin structure and nuclei are shown. Bars, 100 μm. UT, untreated.

Since these findings suggest that SLFN5 controls pathways involved in cytoskeletal rearrangement, we performed RNA-seq analysis to examine the global effects of SLFN5 knockdown on cellular pathways and to identify putative effector elements. RNA-seq analysis showed that 956 genes were upregulated and 570 genes were downregulated following SLFN5 knockdown (
Fig. 7A
). We determined that cancer-related pathways were the highest-ranked pathways affected by SLFN5 knockdown (
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). Additionally, pathways involved in focal adhesion and cell junctions were also regulated by SLFN5 (
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). Overall, elements of pathways involved in the regulation of cell morphology were found to be the most affected in another independent pathway analysis (
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). Significantly, the matrix metalloproteinase 1 gene (
MMP-1
) and
MMP-13
were among the top dysregulated genes by SLFN5 knockdown in the RNA-seq data (
Fig. 7C
).

Two Democratic candidates for the US presidency, Senator Bernie Sanders and Hillary Clinton, are fighting over the term "progressive". But what does the word really mean?

In a CNN
town hall
on 3 February in New Hampshire Mr Sanders and Mrs. Clinton argued over what the word "progressive" means and who has the right to describe themselves in this way.

Mr Sanders said she's not a liberal when it comes to foreign policy and other issues. She disagreed with him, saying that she's "a progressive who likes to get things done". She added that she was "amused" that he'd "set himself up as the gatekeeper of who gets to be a progressive".

So what is a progressive?

Politicians, activists and others disagree about what the word means. Historians concede that there's no precise definition. Still they say that in general a progressive fits certain criteria.

A progressive is someone who wants to see more economic and social equality - and hopes to see more gains in feminism and gay rights. They're also supportive of social programmes directed by the state - and they'd like social movements have more power in the US.

Within the realm of progressive, however, there are different, warring factions, explains David Greenberg, the author of a book called Republic of Spin: An Inside History of the American Presidency.

One group is dominated by activists from social movements such as Occupy Wall Street and Black Lives Matter, he says, and the other is led by those who belong to the left wing of the Democratic Party (and aren't part of a social movement or cause).

Pretty much all of of these progressives "view politics as a bottom-up progress", says Julian Zelizer, an historian at Princeton, and they support the fight for social change. (Though not everybody is on the streets, clamouring for it.)

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Deep Root's database on the open Amazon S3 storage server highlights the years-long effort by companies to stockpile data about American voters. The database includes information from the 2008, 2012 and 2016 presidential campaigns. The 2016 files were not as comprehensive as preceding years.

The leaked data included posts scraped from Reddit. The text from those posts could be used for training computers to recognize language sentiment or to watch specific subreddits to see how people were feeling about political topics.

Some of the exposed information, like voter registration, is public record, but states have different ways of letting people access it and rules on how it can be used.

This is the third time Vickery has found a huge portion of the national voter registration database leaked online. Amazon buckets -- where data is stored -- are private by default. Someone would have to configure the bucket to be public in order to be exposed.

What's also unknown is whether Vickery was the first person to notice the exposed information.

If a bad guy accessed that data, it could be used for stealing someone's identity, stalking individuals or used as leverage for social engineering purposes (that is, tricking something like a phone company into giving someone else your data).

"There's a lot of people hunting for publicly exposed buckets for nefarious purposes," he said.